Cone vision must function over a wide range of light intensities and a broad span of the electromagnetic spectrum. The ability to perform under such diverse conditions arises in large part from the unique set of phototransduction proteins found in cone cells. Light responses initiate with the absorption of photons by the visual pigments (cone opsins). The long-term goals of this project are to understand how the cone opsin family has attained absorption maxima from 350 nm to 650 nm while achieving the rapid photobleaching and regeneration properties that play a large role in shaping the light responses of cones. This proposal will combine molecular, biochemical and biophysical approaches to investigate the molecular mechanisms underlying the function of cone visual pigments. The short wavelength-sensitive (SWS1) visual pigments will be purified from transfected COS cells in order to investigate how particular amino acid residues in the transmembrane helical segments and the extracellular domain regulate the absorbance properties. The conformational changes that occur following isomerization of 11-cis retinal will be studied by low temperature UV/visible spectroscopy and site-directed spin labeling in conjunction with EPR spectroscopy. The amino acids that regulate the formation and stability of the light-activated conformation (R*) that interacts with the cone phototransduction proteins will be studied in conjunction with molecular modeling. Comparative in vitro biochemical studies will investigate the differences between rod and cone signaling, both activation and recovery phases. These experiments will allow a us to address in a comprehensive way several long standing issues about visual pigment behavior: mechanisms controlling spectral tuning, noise and light-activation. Moreover, the characterization of SWS1 functional regions will open the way to study pigment regeneration and folding that may be important in understanding the development and long-term health of cone photoreceptors. [unreadable] [unreadable] [unreadable]